Droplet generating method

ABSTRACT

A droplet generating method includes the steps of providing a micro-pipe having an outlet end; providing a liquid driving device to generate a flow of a first liquid; locating and positioning the micro-pipe which extends along a vertical longitudinal axis; connecting the liquid driving device with the micro-pipe so that the first liquid flows and is emitted out from the outlet end; providing a container, which is positioned at least in-part below the micro-pipe and adapted to contain a second liquid including a liquid surface disposed at a position located between a highest and a lowest positions; and either vertically or horizontally vibrating the micro-pipe, and thereby forming a plurality of droplets of the first liquid emitted from the outlet end at a position below the liquid surface of the second liquid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/576,613, filed on Sep. 19, 2019, now issued as U.S. Pat. No.11,066,695, which is a divisional of U.S. patent application Ser. No.15/598,201 filed on May 17, 2017, now issued as U.S. Pat. No.10,435,737, which is a continuation under 35 U.S.C. § 120 ofinternational patent application PCT/CN2015/077621 filed on Apr. 28,2015, which claims priority to Chinese Application Serial Nos.201410655191.5 and 201410655309.4 filed on Nov. 17, 2014 respectively,the contents of which are also hereby incorporated by reference.

FIELD

The present disclosure relates to manipulation of microliquid, andespecially to droplet generating apparatuses, systems, and methods.

BACKGROUND

Precise and accurate manipulation of nanolilter liquid and microliterliquid is very important to modern engineering science, physicalscience, chemical science, material science, pharmaceutical science,micromachining technology, and widely used in biochemical analysis,environment monitoring, medicine, clinical detection, drug screening,and nanomaterial synthesis and preparation technology. Generatingindependent water-in-oil or oil-in-water microdroplets is very importantto the microliquid manipulation. Multitudinous micro-reactions andmicro-screenings can be realized base on the microdroplets.Multitudinous and uniform microdroplets can be prepared by emulsionpolymerization (referring to Bovey F A et al., Emulsion polymerization,New York: Interscience publishers, 1955, 1-22), membrane emulsification(referring to Nakahsima T et al., Membrane Emulsification by microporousglass, Key Engineering Mater, 1991, 513:61-61), and spay emulsification(referring to Liu. Y et al., Mixing in a multi-inlet vortex mixter(MIVM) for flash nano-precipition, Chemical Engineering Science, 2008,63(11):2892-2842). The above methods are mainly applied in microspherepreparation and drug carrier preparation. However, volumes of themicrodroplets cannot be precisely and accurately controlled. Therefore,these methods are not suitable for a microreactor or complicatedbiochemical reaction which require precise and accurate control of thevolumes of the microdroplets.

Droplet generating method based on microfluidics (referring to The S Yet al., Droplet microfluidics, Lab on a Chip, 2008, 8(2):198-220) hasbeen developed rapidly in recent years. A microdroplet can be generatedin a microchannel of a microfluidic chip based on an unstable interfacebetween a dispersion phase and a continuous phase when they meet in themicrochannel. Through different designs of the microfluidic channel,uniform microdroplets can be generated, merged, reacted, and screened.However, the volumes of the microdroplets are limited by the structureof the microchannel and a surface feature modification of themicrochannel. In addition, the microdroplets generated in themicrochannels must be transferred to a storage container by specificdevice and method, which increases a difficulty to locate, extract, andanalyze the microdroplets.

A simple method used to generate the microdroplet, comprises ejecting orspraying a liquid into a microwell or spotting a liquid on a substrateby a capillary micro-pipe or capillary, for short. However, when themicrodroplet is released from the capillary, it is difficult toprecisely control quantity of microdroplets due to a surface tensionbetween the liquids inside and outside the capillary, and an adhesionforce between the microdroplet and an orifice of the capillary. Toovercome the surface tension, piezoelectric ceramics, thermal expansion,high voltage electronic injection, and ultrasound are used to increase akinetic energy of the microdroplet. To decrease the adhesion force, thestructure of an outlet end of the capillary is modified, and the surfaceof the capillary is coated or silanized. However, complicated andexpensive liquid driving devices are used in these methods, and thevolumes of the microdroplets are difficult to control directly ordefined.

Analysis technology based on digital single molecules and single cellshas been developed in recent years. Uniform microdroplets are idealcarriers of the single molecules and the single cells used inquantitative reaction and analysis. A digital nucleic acid moleculeamplification technology (e.g. digital polymerase chain reaction, dPCR)is a representative digital nucleic acid quantitative analysistechnology, wherein nucleic acid molecules in a sample solution aredistributed to a plurality of micro-reaction systems according to thePoisson distribution, each micro-reaction system substantially compriseseither one nucleic acid molecule or no nucleic acid molecule,independent amplified reaction is carried out in each micro-reactionsystem, and the nucleic acid molecules are quantified by counting anumber of positive micro-reaction systems. The digital nucleic acidmolecule amplification technology is especially suitable for studyingvariation in counts of gene sequence, such as copy number variation andpoint mutation.

Digital enzyme linked immunosorbent assay (Digital ELISA) technologywhich is similar to the digital nucleic acid quantitative analysistechnology is also developed in recent years (referring to Rissin, D. M.et al., Single-molecule enzyme-linked immunosorbent assay detects serumproteins at subfemtomolar concentrations, Nature Biotechnology, 2010,28, 595-599), wherein microspheres containing immune antibodies are putinto a femtoliter microwell array, single target protein molecules areselectively combined with the immune antibodies, the microwell array isimaged by enzyme-linked immunoassay, and the single target proteinmolecules are quantified by counting a number of microwells whosefluorescence signal is amplified.

Digital single cell analysis is one of the hottest fields in modernbiology. The digital single cell analysis is an important method todetect cell heterogeneity which is difficult to be found by conventionalmethod, analyze compositions of cell subset, find low-probability cellmutation, and explore uncultured microorganisms. The single cell is tootiny to be manipulated. The single cell of bacteria, fungus, plants andanimals can be enveloped in the microdroplet with a diameter rangingfrom several tens of microns to several hundreds of microns. Themicrodroplet can supply a micro-environment for the single cell which isconvenient to be manipulated. The extracellular materials secreted bythe single cell can be gathered quickly in picoliter or nanolitermicrodroplets. In addition, multitudinous single cell cultures,enzymatic activity analysis of the single cell, single cell analysis,genome amplification of the single cell, and transcriptome amplificationof the single cell can be realized in the microdroplet.

After a molecule solution or a cell solution is mixed with reactants,the mixture can be distributed into a plurality of microsystems. Asingle molecule or a single cell can be reacted, grown, and amplified ineach microsystem, after which an array detection and a digital analysiscan be applied to the plurality of microsystems, which improvesreliability and sensitivity of biological detection and clinicaldiagnosis, and has a wide application prospect.

SUMMARY

A simple and low-cost droplet generating technology based on amicro-pipe is provided, by which a nanoliter liquid or a microliterliquid can be manipulated to generate multitudinous droplets quickly,precisely, and accurately, and the quantity of the liquid can beadjusted and controlled freely.

Uniform droplets with controllable size can be generated, whichincreases precision and efficiency of microreactions of the droplets,and provides an application basis of the droplets in biology andchemistry.

According to one aspect of the present disclosure, a droplet generatingmethod, comprising:

providing a micro-pipe for dispensing a first liquid and a containercontaining a second liquid, wherein the first liquid is immiscible withthe second liquid;

providing a moving and locating device for positioning the micro-pipeover the container;

providing a liquid driving device connecting to the micro-pipe through aconnecting tube for driving the first liquid through the micro-pipe andout from an outlet end of the micro-pipe;

providing a vibrating equipment connected to the micro-pipe forvibrating the micro-pipe;

forming a relative periodic vibration between the micro-pipe and thecontainer so that the outlet end of the micro-pipe is displaced to touchthe second liquid in the container during a relative periodic vibration;and

dispensing the first liquid in the micro-pipe out from the outlet end ofthe micro-pipe during the relative periodic vibration to generate aplurality of droplets of the first liquid in the second liquid which isinduced by a force of the second liquid imposed on the first liquid atthe outlet end.

According to another aspect of the present disclosure, a dropletgenerating method comprising:

providing a micro-pipe having an outlet end;

providing a liquid driving device to generate a flow of a first liquid;locating and positioning the micro-pipe which extends along a verticallongitudinal axis;

connecting the liquid driving device with the micro-pipe so that thefirst liquid flows and is emitted out from the outlet end;

providing a container, which is positioned at least in-part below themicro-pipe and adapted to contain a second liquid including a liquidsurface disposed at a position located between a highest and a lowestpositions; and

vertically vibrating the micro-pipe along the longitudinal axis betweena highest position and a lowest position, and thereby forming aplurality of droplets of the first liquid emitted from the outlet end.

According to another aspect of the present disclosure, a dropletgenerating method comprising:

providing a micro-pipe having an outlet end;

providing a liquid driving device which is connected with the micro-pipeto generate a flow of a first liquid;

locating and positioning the micro-pipe which extends along a verticallongitudinal axis;

connecting the liquid driving device with the micro-pipe so that thefirst liquid flows and is emitted out from the outlet end;

providing a container, which is positioned at least in-part below themicro-pipe and adapted to contain a second liquid including a liquidsurface, the outlet end of the micro-pipe is under the liquid surface ofthe second liquid; and

horizontally vibrating the micro-pipe with its outlet end swingingbetween a first position and a second position, and thereby forming aplurality of droplets of the first liquid emitted from the outlet end.

According to one aspect of the present disclosure, a droplet generatingapparatus is disclosed. The droplet generating apparatus comprises aliquid driving unit and a vibrating equipment. The liquid driving unitcan comprise a liquid driving device configured to inject a first liquidinto a micro-pipe connected with the liquid driving device. Thevibrating equipment is configured to form a relative motion between themicro-pipe and a container containing a second liquid at a specificfrequency and a vibration amplitude to have the first liquid flowed outfrom an outlet end of the micro-pipe detached from the micro-pipe toform droplets in the second liquid.

The first liquid attached on the outlet end of the micro-pipe can bedetached from the micro-pipe by a fluid shear force of the secondliquid.

In one embodiment, the relative motion is that the micro-pipe moves backand forth (e.g., vibrates) along a longitudinal axis thereof withrespect to the container, and the outlet end of the micro-pipe canrepeatedly go across a liquid surface of the second liquid (which is aninterface between the second liquid and air, or an interface between thesecond liquid and a third liquid), using a surface tension and a fluidshear force generated during the crossing of the liquid surface toovercome a surface force between the first liquid inside and outside themicro-pipe and an adhesion force between the first liquid on the outletend of the micro-pipe, thereby detaching the first liquid on the outletend from the micro-pipe smoothly. One droplet of the first liquid isformed per cycle of the relative vibration. In one embodiment, themicro-pipe is disposed vertically, and vibrated vertically by thevibrating equipment, and an opening of the container can be defined onan upper side of the container. In another embodiment, the micro-pipe isdisposed horizontally, and vibrated horizontally by the vibratingequipment, and the opening of the container can be defined on a side ofthe container.

In one embodiment, the micro-pipe can be vibrated along a directionperpendicular to the longitudinal axis thereof with respect to thecontainer, and the outlet end of the micro-pipe can swing under theliquid surface of the second liquid, during which the first liquidattached on the outlet end can be detached from the micro-pipe due tothe fluid shear force of the second liquid, with a generation throughputof one droplet or two droplets per cycle. In one embodiment, themicro-pipe is disposed vertically, and vibrated horizontally by thevibrating equipment. Because the outlet end of the micro-pipe is underliquid surface in the second liquid all the way, the first liquidflowing out the micro-pipe from the outlet end does not contact withexternal environment, thereby avoiding contamination to the droplets.

A vibration frequency of the vibrating equipment can be in a range fromabout 0.1 Hz to about 5000 Hz, such as from about 1 Hz to about 500 Hz,from about 10 Hz to about 250 Hz, from about 30 Hz to about 200 Hz. Inone embodiment, the vibration frequency of the vibrating equipment is 50Hz. A vibration amplitude of the vibrating equipment can be in a rangefrom about 0.5 mm to about 10 mm, such as from about 1 mm to about 5 mm,and from about 2 mm to about 4 mm.

The vibrating equipment and the liquid driving device are not limited inthe present disclosure. The vibrating equipment can be anelectromagnetic vibrating equipment, a piezoelectric ceramic vibratingequipment, or an eccentric rotating mass (ERM) vibration. The liquiddriving device can be a peristaltic pump, a syringe pump, a pressurepump, a pneumatic pump, and an electroosmotic pump.

The micro-pipe can be vibrated up and down to go across the liquidsurface of the second liquid repeatedly. A distance between a farthestposition out from the second liquid of the outlet end and the liquidsurface of the second liquid can be in a range from about 10% to about90% of the vibration amplitude, such as from about 50% to about 80% ofthe vibration amplitude.

The droplet generating apparatus can further comprise a moving andlocating device. The moving and locating device is configured tosubstantially maintain a distance between the farthest position outsidethe second liquid of the outlet end and the liquid surface of the secondliquid unchanged when a position of the liquid surface of the secondliquid is changed, thereby substantially maintaining the generatingconditions of the droplets unchanged to form uniform droplets.

The moving and locating device can directly or indirectly drive themicro-pipe and/or the container to move in a first direction parallelwith a vibration direction of the micro-pipe and/or a second directionperpendicular to the vibration direction of the micro-pipe.

The liquid driving unit can further comprise a connecting tube. Theliquid driving device can be connected to the micro-pipe through theconnecting tube. One single flow channel can be defined in theconnecting tube, or a plurality of flow channels can be defined in theconnecting tube. The connecting tube can be an airtight tube definingthe plurality of flow channels prepared by micromachining ormicropackaging. The connecting tube can be connected to a plurality ofmicro-pipes. The plurality of flow channels can form a flow channelarray. The liquid driving unit can further comprise a plurality ofconnecting tubes. The plurality of connecting tubes can forma connectingtube array. The connecting tube array or the flow channel array cancorrespond to an array of wells on a microplate such a 96-wellmicroplate. Each connecting tube or each flow channel can be connectedto one micro-pipe independently. The droplets can be generated in thearray of wells of the microplate simultaneously. The microplatecontaining the droplets can be used directly in an analysis device or adetection device.

One end of the connecting tube can be fixed to or detachably connectedto the liquid driving device, and the other end of the connecting tubecan be connected to the micro-pipe. A connecting port can be defined onthe end of the connecting tube and connected to the micro-pipe. Themicro-pipe can be connected to the connecting port by a threadedconnection, a clamping connection, an interference fit, or a plugconnection.

The liquid driving unit can comprise a plurality of liquid drivingdevices. The plurality of liquid driving devices can be connected to aplurality of micro-pipes. The first liquid can be injected into theplurality of micro-pipes at different flow rates by the plurality ofliquid driving devices.

The droplet generating apparatus has simple structure and convenientoperation method. Multitudinous quantitative and uniform droplets can begenerated precisely and accurately by the droplet generating apparatus,thereby decreasing cost of experiment, analysis and detection based onmicroliquid. A droplet array can also be formed by the dropletgenerating apparatus quickly, which can be used for digital reaction anddetection based on single molecule or single cell.

According to another aspect of the present disclosure, a dropletgenerating system is disclosed. The droplet generating system comprisesthe droplet generating apparatus, the micro-pipe, and the container. Thedroplet generating apparatus comprises the liquid driving unit and thevibrating equipment. The micro-pipe can be air-tight connected to theliquid driving unit and contain the first liquid. The containercontaining the second liquid is configured to accept droplets of thefirst liquid.

The micro-pipe can be detachably connected to the liquid driving device.The micro-pipe can be made of at least one of glass, quartz, plastic,and stainless steel. The micro-pipe can be disposable to avoidcontamination. In one embodiment, the micro-pipe is made of stainlesssteel with a high temperature resistance and a high pressure resistance.

At least one microchannel can be defined in the micro-pipe. The outletend of the micro-pipe can be tapered or cylindrical. The micro-pipe canbe selected from a capillary, a bundle of capillaries, a capillaryarray, and a microfluidic channel. The capillary can be selected from asingle core capillary or a multi-core capillary. An upper end of thecapillary can be enlarged to form a liquid storage cavity.

A surface of the outlet end of the micro-pipe can have a low surfaceenergy to detach the droplets more smoothly. The surface of the outletend of the micro-pipe can be modified, such as silanized, to decreaseits surface energy.

The container can be in form of a single container, a one-dimensionalarray of containers, or two-dimensional array of containers. The onedimensional array of containers or the two dimensional array ofcontainers can correspond to the capillary array. The container can beselected from 24-well microplate, 96-well microplate, 384-wellmicroplate, and 1536-well microplate. A bottom of the liquid storagechamber can be flat, tapered, rounded, or oval. In one embodiment, thebottom of the container is flat to accept a droplet array.

Any suitable micro-pipe (e.g. capillary) and container (e.g. microplate)can be used in the droplet generating system, thereby decreasing thecost of the droplet generating system. The container containing thedroplets can be directly analyzed and detected, thereby decreasing thecost of the analysis, detection, and reaction based on microliquid.

According to yet another aspect of the present disclosure, a dropletgenerating method is disclosed, comprising: driving a first liquid outfrom an outlet end of a micro-pipe continuously or intermittently, whileforming a relative vibration between the micro-pipe and a containercontaining a second liquid to detach the first liquid on the outlet endof the micro-pipe from the micro-pipe to form droplets in the secondliquid.

The first liquid on the outlet end of the micro-pipe can be detachedfrom the micro-pipe by a fluid shear force of the second liquid.

The micro-pipe can be vibrated along a longitudinal axis thereof withrespect to the container, and the outlet end of the micro-pipe can goacross a liquid surface of the second liquid repeatedly. In oneembodiment, the micro-pipe is disposed vertically, and vibratedvertically by the vibrating equipment. In one embodiment, the micro-pipeis disposed horizontally, and vibrated horizontally by the vibratingequipment.

The micro-pipe can be vibrated along a direction perpendicular to thelongitudinal axis thereof with respect to the container, and the outletend of the micro-pipe can swing under the liquid surface of the secondliquid. In one embodiment, the micro-pipe is disposed vertically, andvibrated horizontally by the vibrating equipment.

A vibration frequency can be in a range from about 0.1 Hz to about 5000Hz, such as from about 1 Hz to about 500 Hz, from about 10 Hz to about250 Hz, from about 30 Hz to about 200 Hz. In one embodiment, thevibration is 50 Hz. A vibration amplitude can be in a range from about0.5 mm to about 10 mm, such as from about 1 mm to about 5 mm, and fromabout 2 mm to about 4 mm. The first liquid can be different from thesecond liquid. The first liquid and the second liquid can be insolublewith each other. In another embodiment, an interfacial reaction can becarried out by the first liquid and the second liquid.

The droplets of the first liquid in the second liquid can be furtherformed into micro-capsules or microspheres by a reaction between thefirst liquid and the second liquid. The first liquid can be an aqueoussolution, such as sample solution, or reactant solution. The secondliquid can be oil solution, such as petroleum oil (e.g. n-tetradecane),vegetable oil, silicone oil, and perfluorinated alkane. A surfactant canbe added to the second liquid to prevent a fusion between the dropletsof the first liquid. The surfactant can be at least one of nonionicsurfactant, cationic surfactant, anionic surfactant, and ampholyticionic surfactant, such as Span®40, Span®80, and Span®83. A volume ratioof the surfactant to the second liquid can in a range from about 0.01%to about 20% (v/v), such as from about 1% to about 10% (v/v).

The materials of the first liquid and the second liquid are not limitedand can be varied to meet actual need.

The droplet generating method can further comprise preparing the firstliquid, preparing the second liquid, adding the first liquid to themicro-pipe, and adding the second liquid to the container.

A surface of the outlet end of the micro-pipe can be modified, such assilanized, to decrease its surface energy to release the droplets moresmoothly.

A third liquid can be covered on the second liquid. The first liquid,the second liquid, and the third liquid can be insoluble with eachother. The third liquid can form a sealing layer or a protecting layeron the liquid surface of the second liquid to prevent the second liquidfrom evaporation or contamination.

In the droplet generating method, an interfacial energy and a fluidshear force at the gas-liquid interface or the liquid-liquid interfacecan be used to overcome a surface tension and adhesion force of theliquid at the outlet end of the micro-pipe when the outlet end of themicro-pipe having the micro-droplet of the first liquid attached thereongoes across the liquid surface of the second liquid to detach themicro-droplet from the outlet end of the micro-pipe smoothly. Or a fluidshear force can be generated when the outlet end of the micro-pipehaving the micro-droplet of the first liquid attached thereon swings inthe second liquid to overcome the surface tension and adhesion force ofthe liquid at the outlet end of the micro-pipe to detach themicro-droplet from the outlet end of the micro-pipe smoothly.

Sizes of the droplets can be controlled by regulating the first liquid.Multitudinous droplets can be generated quickly, precisely, andaccurately. In addition, the droplets with controllable sizes andvolumes can be directly generated in the second liquid, therebyeliminating a contamination and evaporation of the droplets, andsimplifying an extraction and storage of the droplets.

Volumes of the droplets can be controlled by regulating the flow rate ofthe first liquid, the vibration frequency, the vibration amplitude, etc.The droplets with different components and volumes can be successivelygenerated by changing components of the first liquid, by which not onlymultitudinous high-throughput screening of the micro-liquid can berealized, but also ultramicro biochemical reaction and detection withmulti-steps can be realized.

According to yet another aspect of the present disclosure, an analysisapparatus based on single molecule or single cell is disclosed. Theanalysis apparatus is configured to analyze a single molecule or asingle cell in a microsystem, or carry out a reaction by the singlemolecule or the single cell in the microsystem.

The analysis apparatus can comprise the droplet generating apparatus, aprocessing device and/or an analysis device.

A single molecule or a single cell can be contained inside a droplet.The droplet containing the single molecule or the single cell can beapplied in PCR (polymerase chain reaction) analysis, single moleculeenzyme-linked immunosorbent assay, single cell enzyme activity assay,and single cell genomic amplification sequencing.

The first liquid can be a solution containing molecules or cells. Thedroplet array can be formed by the first liquid in the second liquid bythe droplet generating apparatus.

The molecules can be selected from at least one of nucleic acidmolecules, protein molecules, peptide molecules, organic compoundmolecules, and pharmaceutical molecules.

The cells can be selected from at least one of bacterial cells, fungalcells, plant cells, and animal cells.

The processing device can be selected from at least one of PCR device,heating device, cooling device, and temperature control device. In oneembodiment, a platform on which the container is placed can be directlyheated or cooled by the heating device or the cooling device. In oneembodiment, the processing device further comprises a transfer equipmentconfigured to transport the container to the transfer equipment such asthe PCR device.

The container can be in form of a single container, a one-dimensionalarray of containers, or two-dimensional array of containers. A bottom ofthe container can be planar. In one embodiment, the container is amicroplate, and the micro-pipe is a capillary array corresponding to awell array of the microplate.

The analysis device is configured to analyze the single molecule or thesingle cell contained in the droplet. The analysis device can beselected from at least one of microscope, fluorescence detection device,and ultraviolet-visible detection device. The container containing thedroplets can be directly analyzed and detected by the analysis device.

The analysis apparatus is easy to operate and control. Quantitative anduniform droplets can be generated stably, precisely and accurately. Thecosts of the micro-pipe and container are low, thereby decreasing costof the analysis apparatus. The analysis apparatus can be applied broadlyin microsystems.

BRIEF DESCRIPTION OF THE DRAWINGS

Implementations are described by way of example only with reference tothe attached figures.

FIG. 1 is a schematic view of one embodiment of a droplet generatingapparatus.

FIG. 2 is a schematic view of another embodiment of the dropletgenerating apparatus.

FIG. 3 , FIG. 4 , and FIG. 5 are graphs respectively showing differentembodiments of a droplet distribution in a container.

FIG. 6 is a schematic view of one cycle of a droplet generating processin Example 1.

FIG. 7 is a graph showing a flow rate-time curve of water in a capillaryin Example 1.

FIG. 8 is a graph showing distance-time curve between an outlet end ofthe capillary and a liquid surface of a mineral oil in Example 1.

FIG. 9 is a schematic view of one embodiment of a droplet generatingmethod in Example 3.

FIG. 10 is a schematic view of another embodiment of the dropletgenerating method in Example 4.

FIG. 11 is a microscope photo of droplets laid on a bottom of acontainer in Example 5.

FIG. 12 is a microscope photo of droplets laid on the bottom of acontainer in Example 6.

FIG. 13 is a microscope photo of droplets laid on a bottom of acontainer in Example 7.

FIG. 14 is a microscope photo of droplets laid on a bottom of acontainer in Example 8.

FIG. 15 is a graph showing average volume-flow rate curve of Examples 5to 13 of the droplets.

FIG. 16 is a schematic view of a droplet generating process in one cyclein Example 14.

FIG. 17 is a microscope photo of droplets laid on a bottom of acontainer in Example 14.

FIG. 18 is a microscope photo of droplets laid on a bottom of acontainer in Example 15.

FIG. 19 is a microscope photo of droplets laid on a bottom of acontainer in Example 16.

FIG. 20 is a microscope photo of sodium alginate microspheres in Example17.

FIG. 21 is a microscope photo of droplets laid on a bottom of acontainer in Example 18.

FIG. 22 is a schematic view of a droplet generating method inComparative Example 1.

FIG. 23 is a microscope photo of droplets laid on a bottom of acontainer in Comparative Example 1.

FIG. 24 is a schematic view of a droplet generating method inComparative Example 2.

FIG. 25 is a microscope photo of droplets laid on a bottom of acontainer in Comparative Example 2.

FIG. 26 is a graph comparing average diameter of droplets in Example 18and Comparative Examples 1 to 2.

FIG. 27A and FIG. 27B are respectively front view and section view of amicro-pipe in Example 19.

FIG. 28 is a schematic view of a droplet generating system in Example20.

FIG. 29 is a section view of the droplet generating system in Example20.

FIG. 30 is a schematic view of the droplet generating system in Example21.

FIG. 31 is a schematic view of an analysis apparatus based on singlemolecule or singe cell in Example 22.

FIG. 32 is a microscope photo of droplets laid on a bottom of amicrowell of a 96-well microplate in Example 23.

FIG. 33 is a fluorescence microscope of droplets locating in a greenfluorescence channel after an amplified reaction in Example 23.

FIG. 34 is a distribution graph of fluorescent droplets after theamplified reaction in Example 23.

FIG. 35 is a schematic view of droplets with different volumes showingfluorescence in Example 24.

DETAILED DESCRIPTION

A detailed description with the above drawings is made to furtherillustrate the present disclosure.

A droplet generating apparatus and a droplet generating method areprovided in the present disclosure based on a relative movement of amicro-pipe and a container. A first liquid can flow out from an outletend of the micro-pipe quantitatively. During the relative movement, whenthe outlet end of the micro-pipe goes across a liquid surface of asecond liquid contained in the container, an interfacial energy and afluid shear force can be generated; or when the outlet end of themicro-pipe swings in the second liquid, the fluid shear force can begenerated. Under the action of the interfacial energy and the fluidshear force, the first liquid on the outlet end of the micro-pipe canovercome a surface tension and an adhesive force with an orifice of theoutlet end of the micro-pipe, and be released smoothly from themicro-pipe to form a droplet precisely and accurately in the secondliquid. Multitudinous droplets (e.g. droplets containing a biochemicalsample) can be generated quickly, effectively, and precisely through acontinuous flow of the first liquid from the outlet end of themicro-pipe and the high frequency relative movement of the micro-pipeand the container. Furthermore, quantitative droplets with controllablevolume and number can be directly produced in a dispersant liquid (thesecond liquid), thereby eliminating a contamination and evaporation ofthe droplets, and simplifying an extraction and storage of the droplets.

The first liquid is configured to form the droplets. The second liquidis configured to accept or disperse the droplets. The first liquid canbe different from the second liquid. The first liquid and the secondliquid can be soluble with, insoluble with or partially soluble witheach other. An interfacial reaction can be carried out by the firstliquid and the second liquid. The droplets can be liquid droplets formedby the first liquid, or micro-capsules containing the first liquidinside or microspheres formed by the reaction between the first liquidand the second liquid.

The first liquid and the second liquid can be insoluble with each other.In one embodiment, the first liquid can be an aqueous solution, thesecond liquid can be an oily liquid insoluble with the aqueous solution.The second liquid can be at least one of petroleum oil (e.g.n-tetradecane), vegetable oil, silicone oil, and perfluorinated alkane.The droplets can be formed by the aqueous solution. In one embodiment,the first liquid can be a hydrophobic organic phase, the second liquidcan be an oil phase or aqueous phase insoluble with the hydrophobicorganic phase. In one embodiment, the first liquid and the second liquidcan both be aqueous phase insoluble with each other. For example, thefirst liquid can be a dextran aqueous solution, and the second liquidcan be a polyethylene glycol aqueous solution.

An interfacial reaction can be existed between the first liquid and thesecond liquid. In one embodiment, the first liquid can be a sodiumalginate aqueous solution, the second liquid can be a calcium chlorideaqueous solution (e.g. 1% of mass percentage), and the droplets can becalcium alginate gel microspheres.

A third liquid can be covered on the second liquid. The first liquid,the second liquid, and the third liquid can be immiscible with eachother. The micro-pipe can be vibrated up and down to go across theliquid-liquid interface between the second liquid and the third liquidrepeatedly. In one embodiment, the first liquid can be an aqueoussolution, the second liquid can be a perfluorinated oil (e.g. 3Mfluorinert FC40), and the third liquid can be a mineral oil. Theperfluorinated oil can be covered on the mineral oil due to its lowerdensity. The micro-pipe can be vibrated up and down with respect to aninterface between the perfluorinated oil and the mineral oil, and outputthe first liquid. The droplets formed by the aqueous solution can enterinto the perfluorinated oil. The droplets do not sink and can floatunder a liquid surface of the perfluorinated oil due to its lowerdensity.

Referring to FIG. 1 , in one embodiment, the droplet generatingapparatus can comprise a liquid driving device 4 (in FIG. 1 , a syringepump) and a vibrating equipment 3. A micro-pipe 1 can be connected to adownstream end of the liquid driving device 4. A first liquid 5 can befilled in a cavity of the syringe pump 4, a connecting tube 9, and themicro-pipe 1. A second liquid 6 can be contained in a container 2 underthe micro-pipe 1. The micro-pipe 1 can be vibrated up and downvertically by the vibrating equipment 3. The droplet generatingapparatus can further comprise a moving and locating device 10configured to locate the outlet end of the micro-pipe 1 above the secondliquid 6.

The liquid driving device 4 is configured to drive the first liquid 5 inthe micro-pipe 1 continuously or intermittently. The liquid drivingdevice 4 can be connected to an end of the micro-pipe 1. The liquiddriving device 4 can be selected from a peristaltic pump, a syringepump, a pressure pump, a pneumatic pump, and an electroosmotic pump. InFIG. 1 , the liquid driving device 4 is the syringe pump which is moreprecise and can drive the first liquid at a flow rate of nanoliters perminute.

The flow rate of the first liquid depends on an inner diameter of theoutlet end of the micro-pipe 1, a vibration frequency of the micro-pipe1, a volume of each droplet, and properties of the first liquid and thesecond liquid. In the present disclosure, the flow rate of the firstliquid can be substantially equal to a product of the vibrationfrequency of the micro-pipe 1 and a volume of each droplet. In oneembodiment, the vibration frequency of the micro-pipe 1 is 50 Hz, thevolume of each droplet is 50 picoliter (pL), and the flow rate can beabout 50 Hz×50 pL=2.5 nanoliter (nL).

The micro-pipe can have two open ends. One of the two open ends is aninlet end of the micro-pipe 1 and can be connected to the liquid drivingdevice 4, and the other one is the outlet end of the micro-pipe 1. Thefirst liquid can flow into the micro-pipe 1 through the inlet end, andflow out the micro-pipe 1 through the outlet end. The micro-pipe 1 canbe selected from a single core capillary, a multi-core capillary, abundle of capillaries, a capillary array, and a microfluidic channel. Inone embodiment, an upper end of the capillary is enlarged to form aliquid storage cavity (similar to a needle of a syringe). In oneembodiment, the micro-pipe 1 can be the single core capillary or thecapillary array, which is more simple and cost-effective compared withchips used in microfluidics.

The micro-pipe 1 can be disposable. The micro-pipe 1 is usuallydifficult to wash due to its tiny inner diameter. The disposablemicro-pipe 1 can decrease the cost of the droplet generating apparatus.In one embodiment, the micro-pipe 1 can be detachably connected to theliquid driving device 4.

The two open ends of the micro-pipe 1 comprising the inlet and theoutlet end can be both cylindrical. The outlet end of the micro-pipe 1can also be tapered. Sizes of the two open ends, especially a size ofthe outlet end can be in a range from about 1 μm to about 0.5 mm, suchas from about 5 μm to about 0.25 mm. In one embodiment, an innerdiameter of the outlet end can be in a range from about 5 μm to about250 μm, and an outer diameter of the outlet end can be in a range fromabout 10 μm to 500 μm. To generate smaller droplets, the micro-pipe 1having a smaller inner diameter is required. In one embodiment, themicro-pipe 1 with an inner diameter of about 100 μm and an outerdiameter of about 200 μm is stretched to form a tapered outlet end withan inner diameter of about 30 um and an outer radius of about 50 μm. Thespecific structure and size of the micro-pipe 1 are not limited and canbe varied to meet actual need.

A surface of the outlet end of the micro-pipe 1 can have a low surfaceenergy to release the droplets 7 more smoothly, thereby increasing theprecision of volumes of the droplets 7 and improving uniformity of thedroplets 7. The surface of the outlet end of the micro-pipe 1 can bemodified to decrease its surface energy. The surface of the outlet endof the micro-pipe 1 can be coated with a low surface coating orsilanized.

The liquid driving unit can further comprise a connecting tube 9configured to transport the first liquid from the liquid driving device4 to the micro-pipe 1. The liquid driving device 4 can be air-tightconnected to the micro-pipe 1 through the connecting tube 9. One end ofthe connecting tube 9 can be connected to the liquid driving device 4,and the other end of the connecting tube 9 can be connected to themicro-pipe 1. The connecting tube 9 can be made ofpolytetrafluoroethylene or Teflon®, silicon rubber, polyethylene, orpoly vinyl chloride. One single flow channel can be defined in oneconnecting tube 9, or a plurality of flow channels can be defined in oneconnecting tube 9. The connecting tube 9 can also comprise a pluralityof connecting tubes assembled together. The connecting tube 9 can be anairtight tube defining the plurality of flow channels prepared bymicromachining or micropackaging.

The connecting tube 9 can be fixed to or detachably connected to theliquid driving device 4. When the connecting tube 9 contacts the firstliquid during droplets generation, the connecting tube 9 can bedetachably connected to the liquid driving device 4, so that theconnecting tube 9 can be conveniently replaced according to thedifferent type of the first liquid. When the connecting tube 9 cannotcontact the first liquid during the droplets generation, the connectingtube 9 can be fixed to the liquid driving device 4 to ensure connectionbetween the connecting tube 9 and the liquid driving device 4 isair-tight.

A connecting port can be defined on the end of the connecting tube 9connected to the micro-pipe 1. The micro-pipe 1 can be connected withthe connecting port by a threaded connection, a clamping connection, aninterference fit, or a plug connection. In one embodiment, a structureof the outlet end of the micro-pipe 1 and a structure of the connectingport of the connecting tube 9 are mutually complementary, so that themicro-pipe 1 can be connected to the micro-pipe 1 quickly when replacingthe disposable micro-pipe 1. An inner diameter of the connecting tube 9can be matched with the outer diameter of the micro-pipe 1. Themicro-pipe 1 can be directly connected to the connecting port. Aconnecting joint of the micro-pipe 1 and the connecting tube 9 can besealed by an adhesive to prevent leakage of the first liquid.

If the micro-pipe 1 is a single core capillary or a multi-corecapillary, one connecting tube 9 can be used. If the micro-pipe 1 is thecapillary array in which a plurality of capillaries do not contact witheach other, the plurality of connecting tubes each defining one flowchannel can be used, or one connecting tube defining the plurality offlow channels can be used (referring to FIG. 28 to FIG. 30 ). Theplurality of flow channels can be arranged corresponding to a pluralityof containers. In one embodiment, the plurality of flow channels can bearranged corresponding to a pore array of a perforate plate.

The arrangement of the plurality of flow channels can be correspondingto a well array of a microplate in common use such as 96-wellmicroplate, 384-well microplate, and 1536-well microplate, therebydecreasing the cost of the droplet generating apparatus, and increasinguniversality of the generating apparatus. In addition, the dropletgenerating apparatus can be used in combination with other detectiondevices. In one embodiment, the micro-pipe is a chip defining aplurality of microchannels. An inlet opening is defined atone end of themicrochannel. An inner diameter of the inlet opening can be in a rangefrom about 0.5 mm to about 2 mm. The connecting tube 9 can be directlyinserted into the inlet opening of the chip. An outlet opening can bedefined on the other end of the microchannel. The chip can be made of anelastic material (e.g. polydimethyl siloxane). The outer diameter of theconnecting tube can be only slightly larger than the inner diameter ofthe inlet opening, thereby sealing the connecting tube and the inletopening without the adhesive. When the chip is made of a rigid materialsuch as glass and plastic, a joint between the connecting tube and theinlet opening can be sealed by the adhesive.

The vibrating equipment 3 is configured to drive the micro-pipe 1vibrating with respect to the container 2. Referring to FIG. 1 , in oneembodiment, the vibrating equipment 3 can be connected to the connectingtube 9. The micro-pipe 1 can be driven by the vibrating equipment 3 to areciprocating vibration with respect to the container 2, during whichthe outlet end of the micro-pipe 1 goes across the liquid surface of thesecond liquid repeatedly. Referring to FIG. 16 , in another embodiment,the outlet end of the micro-pipe 1 can be driven by the vibratingequipment 3 to a transverse motion under the liquid surface of thesecond liquid.

The vibrating equipment 3 can drive the reciprocating vibration with asmall amplitude and a high vibration frequency. The vibrating equipment3 can be an electromagnetic vibrating equipment, a piezoelectric ceramicvibrating equipment, or an eccentric rotating mass (ERM) vibrationequipment.

The vibration frequency of the vibrating equipment 3 can be fixed,variable, or hierarchical. The vibration frequency of the vibratingequipment 3 can be in a range from about 0.1 Hz to about 5000 Hz, suchas from about 1 Hz to about 500 Hz, from about 10 Hz to about 250 Hz,from about 30 Hz to about 200 Hz. In one embodiment, the vibrationfrequency of the vibrating equipment 3 is 50 Hz.

The amplitude of the vibrating equipment 3 can be adjusted by regulatingan input voltage of the vibrating equipment 3. The input voltage of thevibrating equipment 3 can be regulated by an electromagnetic vibrator, alinear motor, an actuating motor, or a stepping motor. The amplitude canbe in a range from about 0.5 mm to about 10 mm, such as from about 1 mmto about 5 mm, and from about 2 mm to about 4 mm. If the amplitude istoo small, the outlet end of the micro-pipe 1 cannot completely separatefrom the liquid surface of the second liquid when moving away from thecontainer 2. If the amplitude is too large, the precision of the volumesof the droplets can be decreased. The outlet end of the micro-pipe 1 canreach a highest position and a lowest position during the vibration. Theliquid surface of the second liquid can be located between the highestposition and the lowest position. In one embodiment, a distance betweenthe highest position and the liquid surface can be in a range from about5% of the amplitude to about 95% of the amplitude, such as from about50% of the amplitude to about 80% of the amplitude.

If the container 2 is small, the liquid surface of the second liquid canbe elevated while constantly generating the droplets in the secondliquid. The droplet generating apparatus can further comprise a movingand locating device 10. The moving and locating device 10 is configuredto substantially maintain the distance between the highest position ofthe outlet end of the micro-pipe 1 and the liquid surface of the secondliquid when the position of the liquid surface of the second liquid ischanged, thereby substantially maintaining the generating conditions ofthe plurality of droplets to form uniform droplets. The moving andlocating device 10 can be a moving support, which can move in adirection parallel with a vibration direction of the micro-pipe 1. Ifthe micro-pipe 1 is vertically positioned, the moving and locatingdevice 10 can be a vertical lifting support. The moving and locatingdevice 10 can further move in a 2D plane or a 3D space, therebygenerating droplets in each container of the containers array one byone.

In one embodiment, the moving and locating device 10 can directly orindirectly drive the micro-pipe 1 to move. Referring to FIG. 1 , themoving and locating device 10 can be connected to the vibratingequipment 3, and directly or indirectly drive the micro-pipe 1 to movethrough the vibrating equipment 3. In another embodiment, the moving andlocating device 10 can directly or indirectly drive the container 2 tomove. The moving and locating device 10 can drive a support to move, andthe container 2 is disposed on the support. Or, the container 2 can bedirectly disposed on the moving and locating device 10. In anotherembodiment, the moving and locating device 10 can drive both themicro-pipe 1 and the container 2 to move cooperatively and incoordination.

The moving and locating device 10 can have a function of automaticallylocating, for example, by detecting a distance to the liquid surface ofthe second liquid.

The container 2 is configured to contain the second liquid 6, and acceptthe droplets 7 formed by the first liquid 5. The container 2 can furtherconfigured to storage and transfer the droplets 7.

The container 2 can store microliter liquid or nanoliter liquid. Thecontainer 2 can store at least one droplet, that is, the container canstore a single droplet, or a plurality of droplets. The container 2 canbe in form of a single container, a one-dimensional array of containers,or a two-dimensional array of containers. The container 2 can be amicroplate, such as a standard 96-well ELISA microplate, a standard96-well polymerase chain reaction microplate, a 384-well ELISAmicroplate, or a 384-well polymerase chain reaction microplate. Thecontainer array can store multitudinous droplets having specific amountand specific volumes, and the droplets containing specific components.Mixture and reaction of the droplets with the specific components canbetaken in the container array. A bottom of the container 2 can be flat,rounded, or tapered.

Referring to FIG. 1 , in one embodiment, the micro-pipe 1 can bedisposed above the liquid surface of the second liquid. The outlet endof the micro-pipe 1 can face the liquid surface of the second liquid.The distance between the highest position of the outlet end and theliquid surface of the second liquid depends on the amplitude of thevibrating equipment 3.

Referring to FIG. 2 (wherein the liquid driving device 4 is not shown),in another embodiment, the micro-pipe 1 can be disposed at a side of thecontainer 2. The first liquid 5 can be put into the micro-pipe 1, andthe micro-pipe 1 is moved back and forth relative to the second liquid 6in the container 2 to generate the droplet 7 in the container 2.

It is noted that the relative movement between the micro-pipe 1 and thecontainer 2 essentially refers to a relative movement between the outletend of the micro-pipe 1 and the container 2. The vibrating equipment 3can only drive the container 2 to move, the micro-pipe 1 to move, orboth the container 2 and the micro-pipe 1 to move cooperatively and incoordination.

Referring to FIG. 1 , when the vibrating equipment 3 drives themicro-pipe 1 to move down, the micro-pipe 1 can be moved towards theliquid surface of the second liquid from the highest position until theoutlet end or the first liquid on the outlet end enters into the secondliquid. When the vibrating equipment 3 drives the micro-pipe 1 to moveup, the micro-pipe 1 can be moved away from the second liquid until theoutlet end exited out from the second liquid and returns to its originalposition, during which the first liquid can be released from the outletend, and kept in and surrounded by the second liquid to form one dropletdue to a surface tension and a liquid shear force of the liquid surfaceof the second liquid.

In one embodiment, the micro-pipe 1 can be vertically disposed, and theoutlet end of the micro-pipe 1 can be vibrated up and down with respectto the liquid surface of the second liquid. A distance between thehighest position of the outlet end and the liquid surface can be in arange from about 5% of the amplitude to about 95% of the amplitude, suchas from about 50% of the amplitude to about 80% of the amplitude, forexample, from about 0.5 mm to about 2 mm. In one embodiment, themicro-pipe 1 can be vertically disposed, and the outlet end of themicro-pipe 1 can be vibrated horizontally under the liquid surface ofthe second liquid. In one embodiment, the micro-pipe 1 can behorizontally disposed, and the outlet end of the micro-pipe 1 can bevibrated horizontally under the liquid surface of the second liquid.

In one embodiment, the vibrating equipment 3 can be connected to thecontainer 2, and directly drive the container 2 to vibrate with respectto the micro-pipe 1. The container 2 can be vibrated up and down, orback and forth by the vibrating equipment 3 to contact the liquidsurface of the second liquid with the outlet end of the micro-pipe 1,and to accept the droplets formed by the first liquid.

A plurality of droplets with different components and volumes can besuccessively generated by changing components of the first liquid, bywhich not only multitudinous high-throughput screening of themicro-liquid, but also ultramicro biochemical reaction and detectionwith multi-steps can be realized.

The first liquid can be filled with the micro-pipe 1. The first liquidcan be injected into the micro-pipe 1 continuously or intermittently bythe liquid driving device 4. In one embodiment, the first liquid can beinjected into the micro-pipe 1 continuously in coordinating with thevibration of the outlet end of the micro-pipe 1. In one embodiment, thefirst liquid can be injected into the micro-pipe 1 at a predeterminedflow rate for a predetermine time when the micro-pipe 1 reaches apredetermined position.

Referring to FIG. 3 , when a specific gravity of the first liquid isequal to a specific gravity of the second liquid, the droplets formed bythe first liquid can be suspended in the second liquid freely. Referringto FIG. 4 , when the specific gravity of the first liquid is smallerthan the specific gravity of the second liquid, the droplets can floatnear the liquid surface of the second liquid to form a droplet array.Referring to FIG. 5 , when the specific gravity of the first liquid islarger than the specific gravity of the second liquid, the droplets cansink to a bottom of the container 2 to form a tiled droplet array. Byforming the droplet array, location, imaging, detection, extraction, andanalysis of the droplets can be more convenient. A contamination of thedroplets can be avoided when the droplet array formed at the bottom ofthe container 2, and signals for analysis and detection of the dropletscan be transmitted through the bottom of the container 2.

Diameters and volumes of the droplets can be controlled by regulatingthe flow rate of the first liquid in the micro-pipe 1, the volume of thefirst liquid in the micro-pipe 1, the size of the outlet end of themicro-pipe 1, and the vibration frequency and amplitude of themicro-pipe 1. The volumes of the droplets can be controlled in a widerange from about 20 pL to about 10 nL.

In the present disclosure, the micro-pipe 1 can be filled with the firstliquid, or at least the outlet end can be filled with the first liquid.The micro-pipe 1 can be disposed above the liquid surface of the secondliquid in the container, and then moved towards the liquid surface ofthe second liquid until contact with and entering into the secondliquid. When the first liquid flow out from the outlet end of themicro-pipe 1, the micro-pipe 1 can be moved away from the liquid surfaceof the second liquid vertically or horizontally until the outlet end isexiting out from the second liquid, during which the first liquidflowing out from the outlet end of the micro-pipe 1 can be separatedfrom the micro-pipe 1, and form one droplet in the second liquid. Aninterfacial energy and a fluid shear force can be generated when theoutlet end of the micro-pipe 1 goes across the liquid surface of thesecond liquid to overcome a surface tension and an adhesion forcebetween the first liquid and the surface of the outlet end of themicro-pipe 1, so that the first liquid can be released from themicro-pipe 1 smoothly and quantitatively to form the droplets withcontrollable sizes and volumes. Furthermore, by regulating a vibrationfrequency of the micro-pipe 1 in which the first liquid is continuouslyinjected into and flowed out, multitudinous droplets (e.g. dropletscontaining a biochemical sample) with fixed volumes can be generatedquickly, effectively, and precisely. As the droplets can be directlygenerated in a second liquid, an evaporation of the droplets can beavoided, and the extraction and storage of the droplets can besimplified.

An analysis apparatus based on single molecule or single cell comprisingthe droplet generating apparatus is disclosed. The analysis apparatuscan further comprise a processing device and/or an analysis device.

In one embodiment, the processing device can be an amplification deviceused for single molecule (e.g. nucleic acid molecule) amplification. Theamplification device can be selected from a polymerase chain reactiondevice, a temperature control box, a temperature control heating plate,an infrared heater with a temperature control device, or a wind heaterwith a temperature control device.

The amplification device and the droplet generating apparatus can beindependent from each other, or integrated as one apparatus.

In one embodiment, the amplification device and the droplet generatingapparatus are independent from each other, each with their ownrespective advantages. The droplet generating apparatus can be used as amicro pipette for adding trace amounts of reagents for biologicalreaction or chemical reaction. The amplification device can be used tocarry out an amplified reaction. The container wherein the generateddroplets are stored can be transferred to the amplification device. Asupport for supporting the container during droplet generation can beused to transfer the container and the droplets. The support forsupporting the container can be a moving support or a band carrier. Thecontainer and the droplets can be transferred to the amplificationdevice smoothly to prevent the fusing of the droplets induced by avibration of the container.

In another embodiment, the amplification device and the dropletgenerating apparatus are integrated as one apparatus. After the dropletsare generated, the amplified reaction can be carried out directly in thecontainer, thereby avoiding the vibration of the container, andincreasing the use convenience of the analysis apparatus.

The amplification device can comprise a temperature controller. Thetemperature controller is configured to provide a temperaturecorresponding to each reaction step of the amplified reaction.

The analysis apparatus can further comprise a detection device. Thedetection device is configured to collect and process signals of thedroplets after the amplified reaction, and output detection results.

The detection device can comprise a signal collecting device, a signalprocessing device, and an output device. The signal collecting device isconfigured to collect product signals (e.g. fluorescence signals,ultraviolet signals or turbidity signals) after the amplified reaction.The signal processing device can process the product signals accordingto droplet generating conditions such as the flow rate of the firstliquid and the vibration frequency to calculate a quantitative result.The output device is configured to output the detection results.

The signal collecting device, the signal processing device, and theoutput device is not limited in the present disclosure. The detectiondevice can be an optical microscopic imaging system, a fluorescencescanner, or an integrated imaging sensor. In one embodiment, thedetection device is a fluorescence microscopic imaging system, wherein afluorescence phenomenon of the droplets can be observed, and a number oftemplates DNA in reaction liquid of the amplified reaction can beobtained by counting fluorogenic droplets. In one embodiment, thedroplets can be imaged by the optical microscopic imaging system toobtain all signals of the droplets at one time.

The amplified droplets can further be extracted and used for wholegenome sequencing, single nucleotide polymorphism analysis, copy numbervariation, gene site mutation, and multi-gene expression.

The amplification device and the detection device can be independentfrom each other, or integrated as one apparatus.

In the analysis apparatus, the first liquid can comprise targetmolecules or target cells. The first liquid can further comprisereactants for the amplified reaction. The first liquid can be an aqueoussolution. In one embodiment, the first liquid can comprise nucleic acidtemplates, a buffered aqueous solution, deoxyribonucleoside triphosphate(dNTP), a primer, a polymerase, and marking materials of the product ofthe amplified reaction (e.g. fluorescent material). The first liquid canalso be a positive control reaction liquid or a negative controlreaction liquid.

The sample to be detected can be micro-quantity of liquid containing afew nucleic acid molecules. The sample can be micro body fluid (e.g.blood), small amount of epidermis, or small amount of mucosa. The samplecan comprise a few microorganism (e.g. bacteria, virus, or plasmid)whose quantitative analysis can be realized by detecting specific DNAfragment or RNA fragment of chromosome of the microorganism.

The reaction liquid for the amplified reaction can be nucleic acidamplification reaction liquid (DNA amplification reaction liquid) withdeoxyribonucleic acid (DNA) as template, reverse transcription nucleicacid amplification reaction liquid (RNA reverse transcription reactionliquid) with ribonucleic acid as template, or other nucleic acidamplification reaction liquid such as loop-mediated isothermalamplification reaction liquid. The DNA amplification reaction liquid cancomprise deoxyribonucleoside triphosphate, buffer solution, inorganicions, polymerase, primer, and DNA template. The DNA amplificationreaction liquid can further comprise marking material for detection suchas fluorochrome or fluorescence probe. The RNA reverse transcriptionreaction liquid can comprise reverse transcriptase, RNA inhibitor,buffer solution, inorganic ions, primer, and RNA template.

The marking material can be fluorochrome or fluorescence probe which canindicate the DNA amplification. The fluorochrome can be combined withDNA, such as SYBR Green. The fluorescence probe can be oligosaccharideprobe with fluorophore and quenching group, such as TaqMan fluorescenceprobe. The marking material can also be nano fluorescent granules, orlight absorption material.

The reaction liquid can be pre-mixed uniformly in another container suchas a centrifuge tube, and then the marking material can be added to thereaction liquid.

The first liquid can be prepared and diluted before the amplification toensure that the droplets with predetermined volume comprise at most onemolecule to be amplified. That is, each droplet either comprises onemolecule to be amplified, or does not comprise any molecule to beamplified. A number ratio of the molecule to be amplified to thedroplets can be in a range from about 0.01 to about 0.5, or from about0.1 to about 0.3.

The first liquid and the second liquid can be insoluble with each other.In one embodiment, the first liquid can be an aqueous solution, and thesecond liquid can be an oil phase. The second liquid can be anon-volatile liquid with stable chemical property, not react with thefirst liquid, and have no fluorescence interference. The second liquidcan be at least one of mineral oil (e.g. n-tetradecane), vegetable oil,silicon oil, and perfluorinated alkane.

The second liquid can further comprise a surfactant to prevent fusing ofthe droplets. The surfactant can be inert. A volume ratio of thesurfactant to the second liquid can in a range from about 0.01% to about20% (v/v), such as from about 2% to about 10% (v/v). The surfactant canbe at least one of nonionic surfactant, cationic surfactant, anionicsurfactant, and ampholytic ionic surfactant, such as Span®40, Span®80,and Span®83. In one embodiment, the surfactant is 5% (v/v) of Span®80.

The second liquid can supply the fluid shear force for the first liquidto form the droplets, and an environment in which the droplets can bepreserved stably and isolated from external environment duringexplicated reaction, thereby avoiding contamination and volatilizationof the droplets, and increasing precision of the single moleculeanalysis apparatus or the single cell analysis apparatus.

In one embodiment, the specific gravity of the first liquid is largerthan the specific gravity of the second liquid. The droplets can sink tothe bottom of the container and form the droplet array. One layer or aplurality of layers of the droplets can be formed at the bottom of thecontainer. One layer of the droplets is preferred due to detectionconvenience. In one embodiment, the specific gravity of the first liquidis substantially equal to the specific gravity of the second liquid, thedroplets can be dispersed in the second liquid freely. In oneembodiment, the specific gravity of the first liquid is smaller to thespecific gravity of the second liquid, the droplets can float at anupper portion of the second liquid.

Example 1

Referring to FIG. 6 to FIG. 8 , a quartz capillary 1 is previouslysilanized by a dichloro dimethyl silane to make its surface hydrophobic.The capillary 1 has an inner diameter of 100 μm, an outer diameter of300 μm, and a length of 5 cm. An upper end of the capillary 1 isconnected to a syringe pump (Harvard Apparatus, Pico Elite, not shown inFIG. 6 ) through a Teflon® or polytetrafluoroethylene tube with an innerdiameter of 300 μm and an outer diameter of 600 μm, wherein connectionjoints are sealed by an epoxy resin. A syringe (not shown) with a volumeof 10 μL is connected to the syringe pump. The syringe, the Teflon®tube, and the capillary 1 are filled with water 5, and a leakagedetection is taken before a droplet generating. A glass cell with alength of 1 cm, a width of 1 cm, and a height of 5 cm is used as acontainer 2. A mineral oil 6 with a volume of 4 mL is put into thecontainer 2.

The capillary 1 is vertically disposed and fixed on a vibrator capableof vibrating up and down. The capillary 1 is vibrated up and down by thevibrator with a vibration frequency of 0.1 Hz and an amplitude of 10 mm.In one cycle (a vibration period of 10 seconds), at zero second, aninitial position of an outlet end of the capillary 1 is 5 mm above aliquid surface of the mineral oil 6. The capillary 1 is driven by thevibrator move down vertically at a rate of 2 mm/s from the initialposition to enter the mineral oil 6 at a depth of 5 mm. Then thecapillary 1 is immediately moved up vertically at a rate of 2 mm/s untilit is back to the initial position. During the moving, the water 5 isinjected into the capillary 1 continuously at a flow rate of 2 nL/s bythe syringe pump, driven out from the outlet end of the capillary 1, anddetached from the outlet end of the capillary 1 to form one droplet 7with a volume of 20 nL in the mineral oil 6 at the 9th second. Thedroplet 7 sinks to a bottom of the container 2 due to its higherspecific gravity. A plurality of uniform droplets each having a volumeof 20 nL are generated during the vibration.

The flow rate of the water 5 and the moving speed of the capillary 1 canbe increased corresponding to each other to decrease the time forforming the 20 nL droplet.

The injection of the water 5 and the movement of the capillary 1 arecontinuous, thereby increasing efficiency of generating droplets.Multitudinous droplets can be generated conveniently in this example.

Example 2

The method in Example 2 is substantially the same as the method inExample 1, except that the flow rate of the water injected by thesyringe pump is 1 nL/s, 5% (v/v) of Span®80 is pre-added to the mineraloil to prevent fusing of the droplets, and each generated droplet has avolume of 10 nL.

The volume of each droplet can be adjusted by regulating the flow rateof the water.

Example 3

Referring to FIG. 9 , the method in Example 3 is substantially the sameas the method in Example 1, except three parallel capillaries 1 with aspace of 2 mm therebetween are used. The three capillaries arerespectively connected to three syringes through three silicon rubbertube. Each capillary has an inner diameter of 50 μm and an outerdiameter of 155 μm. Each silicon rubber tube has an inner diameter of150 μm and an outer diameter of 600 μm. A volume of each the syringe is50 μL. The three syringes are driven by a syringe pump.

Three droplets are generated simultaneously in one cycle. Multitudinousdroplets can be generated by using the array of capillaries effectivelyand conveniently.

Example 4

Referring to FIG. 10 , the method in Example 4 is substantially the sameas the method in Example 3, except that the three capillaries 1 arereplaced by a microfluidic chip 1 defining an array 2-1 of five parallelmicrochannels 2-2, five inlet 2-3 openings are respectively defined onupper end of the five microchannels, five connecting tubes made ofTeflon are respectively inserted into the five inlet openings, the fivetubes are further respectively connected to five syringes with a volumeof 50 μL, and the five syringes are driven by a syringe pump having fiveflow channel.

The microfluidic chip is made of polydimethylsiloxane, and prepared bysoft lithography. The microfluidic chip has a thickness of 2 mm, a widthof 8.5 mm, and a length of 2 cm. Each microchannel has a depth of 30 μm,a width of 100 μm, and a length of 1 cm. A spacing between adjacentmicrochannels in the five parallel microchannels is 1.5 mm. Eachmicrochannel corresponds to a tapered outlet end. A diameter of eachsample port is 500 μm. Each Teflon® tube has an inner diameter of 300μm, an outer diameter of 600 μm, and a length of 20 cm. Each Teflon®tubes can be inserted into each inlet openings made by elasticpolydimethylsiloxane with a good air tight seal.

Five droplets can be generated simultaneously in one cycle.Multitudinous droplets can be generated by using the array ofmicrochannels effectively and conveniently.

Examples 5 to 8

The methods in Examples 5 to 8 is substantially the same as the methodin Example 1, except that the mineral oil 6 is replaced by a tetradecanecontaining 5% (v/v) of Span®80, the flow rate of the water arerespectively 2.4 μL/min, 4.8 μL/min, 6 μL/min, and 12 μL/min, thevibration frequency is 50 Hz, the amplitude of the capillary is 3 mm,the initial position of the outlet end of the capillary 1 is 2 mm abovea liquid surface of the tetradecane, and the outlet end of the capillary1 enters into the tetradecane at a depth of 1 mm when moving down.

Referring to FIG. 11 to FIG. 14 , an average diameter of the dropletsgenerated in Example 5 is 178 μm, and a volume standard deviation is7.2%. An average diameter of the droplets generated in Example 6 is222.4 μm, an average volume is 5.76 nL, and a volume standard deviationis 6.0%. An average diameter of the droplets generated in Example 7 is229.3 μm, an average volume is 6.31 nL, and a volume standard deviationis 8.9%. An average diameter of the droplets generated in Example 8 is305.8 μm, an average volume is 15.0 nL, and a volume standard deviationis 3.0%.

Examples 9 to 13

The methods in Examples 9 to 13 is substantially the same as the methodin Example 5, except that the flow rates of the water are respectively0.06 μL/min, 0.30 μL/min, 0.60 μL/min, 1.2 μL/min, and 18 μL/min.

Referring to FIG. 15 , the average volume of the droplets is calculatedaccording to the average diameter of the droplets. It can be seen fromFIG. 15 that there is a significant linear correlation between the flowrate of the water and a volume of the droplet. A formula of the linearfitting is: v=0.0685×f+0.1453, R²=0.9997, wherein v is the volume of thedroplet, and f is the flow rate of the water. It can be concluded thatthe volume of the droplet can be controlled precisely by regulating theflow rate of the first liquid.

Example 14

Referring to FIG. 16 , the micro-pipe 1 is vibrated horizontally underthe liquid surface of the first liquid to form the droplets. A capillary1 with an inner diameter of 25 μm and an outer diameter of 50 μm isconnected to a syringe with a volume of 50 μL filled with water by aTeflon® tube. The capillary 1 is inserted vertically under a liquidsurface of the mineral oil, and fixed to a vibrating reed of anelectromagnetic vibrator. The capillary 1 is vibrated horizontally witha vibration frequency of 50 Hz and an amplitude of 2 mm, during whichthe outlet end of the capillary 1 is stayed under the liquid surface ofthe mineral oil. The water is injected to the capillary 1 at a flow rateof 3 μL/min by a syringe pump. Referring to FIG. 17 , the average volumeof the droplets is 2.63 nL, and a volume standard deviation is 5.5%.

Example 15

The method in Example 15 is substantially the same as the method inExample 14, except that the amplitude is 3 mm. Referring to FIG. 18 ,the average volume of the droplets is 1.41 nL, and the volume standarddeviation is 0.68%.

Example 16

The method in Example 16 is substantially the same as the method inExample 14, except that the amplitude is 4 mm. Referring to FIG. 19 ,the average volume of the droplets is 0.47 nL, and the volume standarddeviation is 56.9%.

It can be seen from FIG. 17 to FIG. 19 that sizes and uniformity of thedroplets are influenced by the amplitude of the outlet end of themicro-pipe 1 when the outlet end of the micro-pipe 1 is vibratedhorizontally in the second liquid with a constant vibration frequencyand a constant flow rate of the water. At a flow rate of 3 μL/min of thewater, the droplets generated with an amplitude of 2 mm to 3 mm is moreuniform than the droplets generated with an amplitude of 4 mm.

The size precision of the droplets is influenced by the flow rate of thefirst liquid and an amplitude of the outlet end of the micro-pipe 1 whenthe outlet end of the micro-pipe 1 is vibrated horizontally in thesecond liquid. The droplets can be directly generated in the secondliquid, thereby avoiding contact with air, and eliminating thecontamination and the evaporation of the droplets.

Example 17

Sodium alginate microspheres are prepared, and single cell microbeculture is taken in the sodium alginate microspheres in this example.

The first liquid is a sodium alginate aqueous solution containing 0.9%(w/v) of sodium chloride and 1×10⁸ cell/ml of Escherichia coli cells.The second liquid is a 1% (v/v) of calcium chloride aqueous solution.

A quartz capillary 1 with an inner diameter of 40 μm and an outerdiameter of 100 μm is previously silanized by a dichloro dimethyl silaneto make its surface hydrophobic. The capillary 1 is vertically fixed ona vibrator and vibrated up and down by the vibrator at a vibrationfrequency of 50 Hz with an amplitude of 2.5 mm. During vibration, adistance between an outlet end of the capillary 1 and the liquid surfaceof the second liquid is 2 mm at highest position above the liquidsurface, and 0.5 mm at lowest position under the liquid surface. Thefirst liquid is injected into the capillary 1 at a flow rate of 0.3μL/min to ensure that the capillary 1 is filled with the first liquid.When the first liquid entered into the second liquid along with theoutlet end of the capillary 1, the sodium alginate are crosslinked andsolidified due to calcium ions to form the sodium alginate microspherescontaining Escherichia coli cells, and the sodium alginate microspheresare detached from the outlet end of the capillary 1 due to surfacetension and fluid shear force. Referring to FIG. 20 , a diameter of eachsodium alginate microsphere is 32.2 μm with a standard deviation of8.3%.

After the microspheres are generated, 400 μL of 5% (v/v) of calciumchloride aqueous solution is added to the container to solidify themicrospheres again. The microspheres can be separated from the secondliquid by centrifugation at 3000 rpm for 3 min, and put into a LB(luria-bertani) culture medium. The Escherichia coli cells are culturedat a temperature of 37° C. After the culture, the microspheres areobserved by a microscope. A micro-colony grown by the Escherichia colicells is observed. Multitudinous microorganisms can be separated fromeach other and then be cultured independently by the microspheres.Manipulation, separation, purification, and analysis of the micro-colonyare convenient.

Example 18

The method in Example 18 is substantially the same as the method inExample 1, except that the capillary 1 has an inner diameter of 30 μmand an outer diameter of 50 μm. The second liquid is a tetradecanecontaining 5% (v/v) of Span®80, the first liquid is water.

Comparative Example 1

Referring to FIG. 22 , the method in Comparative Example 1 issubstantially the same as the method in Example 18, except that theoutlet end of the capillary 1 vibrated up and down under a liquidsurface of the tetradecane.

Referring to FIG. 21 and FIG. 23 , it can be seen that the dropletsgenerated in Example 18 are more uniform than the droplets generated inComparative Example 1.

Comparative Example 2

Referring to FIG. 24 , the method in Comparative Example 1 issubstantially the same as the method in Example 18, except that theoutlet end of the capillary 1 vibrated up and down above a liquidsurface of the tetradecane, and do not contact the liquid surface of thetetradecane.

Referring to FIG. 21 and FIG. 25 , it can be seen that the dropletsgenerated in Example 18 are more uniform than the droplets generated inComparative Example 2.

Referring to FIG. 26 , it can be seen more directly that the dropletsgenerated in Example 18 are more uniform compared to ComparativeExamples 1 to 2.

Example 19

Referring to FIG. 27 , a micro-pipe comprising a liquid storage cavityis provided. The micro-pipe can be detachably connected to theconnecting tube quickly. The micro-pipe can comprise a straight tube 1-1and a liquid storage cavity 1-2 defined on an upper end of the straighttube 1-1. The structure of the micro-pipe is similar to a disposableneedle of a syringe. The micro-pipe can be made at least one ofstainless steel, glass, and quartz. The micro-pipe can be one piece. Aninner diameter of the straight tube 1-1 can be in a range from about 50μm to about 200 μm, such as about 60 μm. An outer diameter of thestraight tube 1-1 can be in a range from about 200 μm to about 500 μm,such as about 200 μm. A length of the straight tube 1-1 can be in arange from about 0.5 cm to about 10 cm, such as about 2 cm. The liquidstorage cavity 1-2 is configured to store the first liquid 5 containingsamples. An mineral oil 8 can be disposed on the first liquid 5 toprevent evaporation of the first liquid. A bottom of the liquid storagecavity 1-2 can be tapered. The upper end of the liquid storage cavity1-2 is configured to detachably connect to the connecting tube, and canhave an inner diameter corresponding to the outer diameter of theconnecting tube. An inner diameter of the liquid storage cavity 1-2 canbe in a range from about 0.5 mm to about 10 mm, such as about 4 mm. Avolume of the liquid storage cavity 1-2 can be in a range from about 10μL to about 1000 μL, such as about 150 μL.

Example 20

Referring to FIG. 28 and FIG. 29 , the micro-pipe 1 comprises aplurality of micro-pipes forming a two-dimensional array. Eachmicro-pipe of Example 20 is the same as the Example 19. Thetwo-dimensional array is formed by ninety-six micro-pipes arranged innine rows and twelve columns. The connecting tube 9 defines a tubularinlet 9-1, a rectangular cavity 9-2, and a plurality of flow channels9-3. The tubular inlet 9-1 communicates with the plurality of flowchannels 9-3 through the rectangular cavity 9-2. The plurality of flowchannels 9-3 are parallel with each other, and communicate with therectangular cavity 9-2 respectively. A bottom end of each flow channel9-3 is extended to connect with one micro-pipe. The distribution of theplurality of flow channels 9-3 corresponds to the two-dimensional arrayand a container 2 which is a 96 well microplate. A liquid driving device4 can connect to the common inlet 2-3 of the array of micro-pipes via aconnecting tubing 9. The inner channel 2-4 of the common inlet 2-3 isconnected with all flow channels 9-3 above the micro-pipes 1. Each flowchannel 9-3 has an inner radius of 4 mm and an outer radius of 6 mm. Aspacing of the plurality of flow channels 9-3 is 9 mm. The rectangularcavity 9-2 has a length of 12 cm, a width of 8 cm, and a height of 5 mm.

100 μL of a mineral oil is added to each pore of the 96 well microplate.100 μL of sample liquid is injected into each flow channel firstly. Eachmicro-pipe is aligned to each pore of the 96 well microplate. A distancebetween an outlet end of each micro-pipe and the liquid surface of themineral oil is 2 mm above the liquid surface.

The connecting tube 9 is vibrated by a vibrator at a vibration frequencyof 50 Hz and with an amplitude of 3 mm. The flow rate of the sampleliquid is 100 nL/s. Fifty droplets each having a volume of 2 nL aregenerated in each pore in each second. The droplets generating can berepeated only be replacing another 96 well microplate.

The liquid driving device can apply an air pressure on a liquid surfaceof the sample liquid stored in the liquid storage cavity 1-2 to drivethe sample liquid contained in the micro-pipe flow, thereby preventpollution of the sample liquid to the liquid driving device.

Example 21

Referring to FIG. 30 , the method in Example 21 is substantially thesame as the method in Example 20, except that the micro-pipe is aone-dimensional array formed by eight micro-pipes. Droplets can begenerated in one row of pores of the 96 well microplate simultaneously.A liquid driving device 4 can connect to the common inlet 2-3 of thearray of micro-pipes via a connecting tubing 9. The one-dimensionalarray can be moved from one row to another row of pores of the 96 wellmicroplate by the moving and locating device (not shown) to generatedroplets in all pores of the 96 well microplate.

Example 22

Referring to FIG. 31 , the droplet generating device is used foramplification of singe molecule. The liquid driving device 4 (a syringepump) is connected to a capillary 1 to form an airtight connection. Afirst liquid 5 containing a sample is filled with a cavity of thesyringe pump and the capillary 1, and flows out an outlet end of thecapillary 1 driven by the syringe pump. The capillary 1 is fixed to avibrating equipment 3 through a clamp, and vibrated up and down alongits vertical axis by the vibrating equipment 3. The outlet end of thecapillary 1 is vibrated up and down with respect to a liquid surface ofthe second liquid contained in the container 2. The first liquid and thesecond liquid are insoluble with each other. When the outlet end of thecapillary 1 goes across the liquid surface, the first liquid flowing outfrom the micro-pipe is released from the outlet end of the micro-pipe toform one droplet. The droplets are arrayed at the bottom of thecontainer 2 due to its larger specific gravity compared with the secondliquid. It can be seen from FIG. 31 that some droplets 7-1 each containsone molecule, and some droplets 7-2 each contains none droplet. Thecontainer 2 can be put in a nucleic acid amplification device to carryout an amplified reaction. The droplets 7-1′ contain amplified nucleicacid molecule.

Example 23

300 ng/μL of λ-DNA (bought from TAKARA corporation, extracted from λphage) is diluted by a factor of about 10⁶, put into a PCR (PolymeraseChain Reaction) device, and denaturalized at 95° C. for 10 min.

Preparation of LAMP (Loop-mediated isothermal amplification) reactionliquid: supplying 12.5 μL of buffer liquid comprising 40 mmoL/L ofTris-HCl, 20 mmol/L of KCl, 16 mmol/L of MgSO₄, 20 mmol/L of (NH₄)₂SO₄,0.2% of Tween®20, 1.6 mol/L of glycine betaine, 2.8 mmol of dNTPs(deoxy-ribonucleoside triphosphate); supplying primers comprising 40pmol of FIP, 40 pmol of BIP, 20 pmol of Loop-F, 20 pmol of Loop-B, 5pmol of F3, and 5 pmol of B3; adding the primers, 1 μL of Bst DNApolymerase solution, 1 μL of Calcein, 1 μL of λ-DNA, 2 mg/mL of bovineserum albumin solution, and sterile water to the buffer liquid to form a25 μL of LAMP reaction liquid.

The gene sequences of the above primer are respectively listed as:

Primer name Primer sequence (5’-3’) FIPCAGCA TCCCT TTCGG CATAC CAGGT GGCAA GGGTA ATGAGG (SEQ ID NO: 1) BIPGGAGG TTGAA GAACT GCGGC AGTCG ATGGC GTTCG TACTC (SEQ ID NO: 2) F3GAATG CCCGT TCTGC GAG (SEQ ID NO: 3) B3TTCAG TTCCT GTGCG TCG (SEQ ID NO: 4) Loop-FGGCGG CAGAG TCATA AAGCA (SEQ ID NO: 5) Loop-BGGCAG ATCTC CAGCC AGGAA CTA (SEQ ID NO: 6)

150 μL of mineral oil comprising a surfactant is added to a pore of a 96well microplate with a pore volume of 200 μL and an inner diameter of 8mm. A capillary with a length of 5 cm, an inner diameter of 30 μm, andan outer diameter of 60 μm is connected to a syringe filled with 50 μLof mineral oil through a Teflon tube with an inner diameter of 300 μm,wherein connection joints are sealed by epoxy resin. The 50 μL ofmineral oil contained in the syringe is injected into the Teflon® tubeand the capillary by a syringe pump to expel air out the Teflon tube andthe capillary.

The LAMP reaction liquid is sucked into the Teflon tube and thecapillary by the syringe. The connection joint of the Teflon tube andthe capillary is fixed on a vibrating reed of a vibrator. The capillaryis hung upon the liquid surface of the mineral oil contained in the poreof the 96 well microplate. A distance between the outlet end of thecapillary and the liquid surface is 1 mm. The capillary is vibrated upand down by the vibrator at a vibration frequency of 50 Hz and with anamplitude of 3 mm. The LAMP reaction liquid is injected into thecapillary at a flow rate of 100 nL/s with a total volume of 4 μL. After4 μL of the LAMP reaction liquid is injected, 90% of the bottom of thepore of the 96 well microplate is lie with the generated droplets.

The 96 well microplate is put into the PCR device. The droplets in the96 well microplate are amplified simultaneously at 63° C. for 1 hours.

Referring to FIG. 32 to FIG. 34 , a total number of the generateddroplets is 1776. A number of the droplets whose fluorescence intensityis higher than threshold value of 470 is 1105 which is 62% of the totalnumber of the generated droplets, and corresponding to a number ofinitial DNA templates.

Example 24

The method in Example 24 is substantially the same as the method inExample 23, except that the flow rates of the LAMP reaction liquid arerespectively 200 nL/s, 80 nL/s and 10 nL/s to obtain the droplets eachhaving a volume of 4 nL, 1.7 nL, and 0.25 nL.

Referring to FIG. 35 , it can be seen that the average number of DNAmolecule contained in the droplets are decreased along with the averagevolume of the droplets. The blacked droplet indicates that an amplifiedreaction are carried out inside.

Finally, it is to be understood that the above embodiments are intendedto illustrate rather than limit the present disclosure. Variations maybe made to the embodiments without departing from the spirit of thepresent disclosure as claimed. Elements associated with any of the aboveembodiments are envisioned to be associated with any other embodiments.The above-described embodiments illustrate the scope of the presentdisclosure but do not restrict the scope of the present disclosure.

What is claimed is:
 1. A droplet generating method comprising:generating a flow of a first liquid contained in a liquid drivingdevice; directing the flow of the first liquid into a micro-pipeincluding a vertical portion extending along a longitudinal axis, theportion having an outlet end; passing the first liquid from the outletend of the micro-pipe; directing the first liquid from the micro-pipeinto a container positioned at least in-part below the outlet end, thecontainer holding a second liquid including a liquid surface disposed ata position located between a highest position and a lowest position; andforming a plurality of droplets of the first liquid emitted from theoutlet end by vertically vibrating the micro-pipe along the longitudinalaxis between the highest position and the lowest position.
 2. Thedroplet generating method according to claim 1, wherein the containercomprises a plurality of containers, the micro-pipe comprises aplurality of micro-pipes, and the outlet end of each of the plurality ofmicro-pipes is positioned in a corresponding one of the plurality ofcontainers.
 3. The droplet generating method according to claim 2,further comprising: a connecting tube extending between the liquiddriving device and the plurality of micro-pipes.
 4. The dropletgenerating method according to claim 1, wherein the plurality ofdroplets of the first liquid is immiscible with the second liquid. 5.The droplet generating method according to claim 1, wherein forming theplurality of droplets includes forming micro-capsules containing thefirst liquid.
 6. The droplet generating method according to claim 1,wherein forming the plurality of droplets includes generating a reactionbetween the first liquid and the second liquid forming microspheres ofthe first liquid.
 7. A droplet generating method comprising: providing aliquid driving device containing a first liquid; connecting the liquiddriving device to a micro-pipe having a portion extending along alongitudinal axis, the portion including an outlet end; generating aflow of the first liquid into the micro-pipe with the liquid drivingdevice; passing the first liquid from the outlet end of the micro-pipe;directing the first liquid from the micro-pipe into a container, whichis positioned at least in-part below the outlet end, the containerholding a second liquid including a liquid surface, the outlet end ofthe micro-pipe being under the liquid surface of the second liquid; andforming a plurality of droplets of the first liquid by horizontallyvibrating the micro-pipe causing the outlet end to transition between afirst position and a second position.
 8. The droplet generating methodaccording to claim 7, wherein the container comprises a plurality ofcontainers, the micro-pipe comprises a plurality of micro-pipes, and theoutlet end of each of the plurality of micro-pipes is positioned in acorresponding one of the plurality of containers.
 9. The dropletgenerating method according to claim 7, wherein the first liquid isimmiscible with the second liquid.
 10. The droplet generating methodaccording to claim 7, wherein forming the plurality of droplets includesforming micro-capsules containing the first liquid.
 11. The dropletgenerating method according to claim 7, wherein forming the plurality ofdroplets includes generating a reaction between the first liquid and thesecond liquid forming microspheres of the first liquid.